Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

Abstract

Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely Ser³⁴⁶, Ser³⁵¹, Thr³⁶², and Ser³⁶³, and proceeded with a temporal hierarchy, with Ser³⁴⁶ as the first site of phosphorylation. G protein–coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which in turn facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein–dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

Introduction

The nociceptin/orphanin FQ peptide receptor (NOP receptor; NOPR) is the fourth-discovered member of the opioid receptor family and is still the least characterized member (1–4). An endogenous neuropeptide identified from rat and porcine brain extracts was found to activate the NOP receptor by inhibiting cyclic adenosine monophosphate (cAMP) accumulation in transfected cells and was named nociceptin/orphanin FQ (N/OFQ) (5, 6). Through coupling to Gα_i/Gα₀ proteins, NOP receptor activation by N/OFQ leads to inhibition of adenylate cyclase and calcium channels (N-, L- and P/Q-type), as well as the activation of G protein-coupled inwardly rectifying potassium (GIRK) channels (3, 6–14). In addition, various proteins such as protein kinase C (PKC), phospholipase A2 (PLA2), extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) are also activated by NOP receptors (12, 15–20). The NOP receptor is widely expressed throughout the brain, spinal cord and dorsal root ganglia (DRG) (21–26) and is involved in the regulation of important physiological processes, such as learning and memory, emotion, food intake, locomotion, respiration, and immune defense (6, 27–35). NOP receptors are also involved in renal, cardiovascular and gastrointestinal functions, as well as pain perception, addiction and tolerance development (36–41).

The NOP receptor is under active investigation as a therapeutic drug target for many indications. Non-peptidic, small-molecule NOP receptor agonists have been investigated preclinically as anxiolytics and substance abuse medications, and clinically as potential anti-tussives (42). Being in the opioid receptor family, the NOP receptor has been shown to modulate the classical mu opioid (MOP) receptor pharmacology in pain and reward pathways. Intracerebroventricular N/OFQ administration can block morphine, cocaine, alcohol, or methamphetamine induced rewarding effects, as well as increases in extracellular dopamine in mesolimbic pathways (43–48). Novel analgesics with bifunctional activity targeting the MOP and NOP receptor have been developed, one of which is in clinical trials. (49–61). Cebranopadol, the first bifunctional MOP/NOP receptor agonist to reach Phase III clinical trials for acute and chronic pain, has potent anti-nociceptive activity in rodent pain models and is ~1000 times more potent and longer-lasting than morphine in chronic pain assays (59, 61). Cebranopadol exhibited a reduced side-effect profile including development of tolerance, motor deficits or respiratory depression compared with classical MOP receptor agonists (58, 60–65). AT-121, a MOP/NOP receptor bifunctional ligand with partial agonist activity at both receptors, was reported to show morphine-comparable analgesic effects in nonhuman primates, and no side effects such as respiratory depression, abuse potential and physical dependence (66).

Given the global opioid epidemic, alternatives and approaches to decrease the side-effects of classical opioid-based drugs have focused on biased ligands which preferentially activate the G protein-dependent signaling cascades over the G protein-independent signaling, such as arrestin-mediated signaling (67–70). Such differential activation was found to lead to a dissociation of analgesic effects from adverse effects such as respiratory depression, gastrointestinal effects and tolerance, the former considered a G protein-mediated effect (analgesic efficacy) and the latter (undesired effects) due to arrestin recruitment. Such correlations form the fundamental basis for the development of the biased MOP receptor agonist TRV130 that recently completed Phase III clinical (70–75). TRV130 is “biased” for the G protein-mediated pathway over the arrestin pathway, and shows markedly reduced receptor internalization and arrestin recruitment (76, 77).

The clinical use of drugs targeting NOP receptors requires a profound understanding of the NOP receptor system at the physiological and molecular levels. However, much less is understood about NOP receptor signaling after ligand binding. A report by Chang et al. (78) showed that various NOP receptor agonists had differential efficacy in activating G-protein-dependent cAMP inhibition compared to G protein-independent arrestin recruitment, leading to functional selectivity or “signaling bias”. Similarly, other studies demonstrated that NOP receptor partial agonist AT-090 showed arrestin-biased functional selectivity in recombinant cells (79). The detailed molecular events occurring after ligand binding that lead to signaling bias at the NOP receptor are not as yet completely unraveled. In particular, key events in the regulation of GPCRs such as receptor phosphorylation, internalization and desensitization that have been characterized in great detail for the classical opioid GPCRs are not well understood for the NOP receptor (80–86). For example, in the closely-related MOP receptor, a cluster of four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely Ser³⁷⁵, Thr³⁷⁰, Thr³⁷⁶ and Thr³⁷⁹ are phosphorylated by GPCR kinases 2 and 3 (GRK2/3) as well as GRK5 in an agonist-selective and hierarchical manner (87–89). Our own studies with MOP receptor show that its phosphorylation and internalization occurs in an agonist-specific and time-dependent manner and is determined by a 10-residue sequence in the carboxyl-terminal tail of the receptor. Ser³⁷⁵, present in the middle of this sequence, is phosphorylated by many opioids. Morphine, oxycodone and buprenorphine stimulate phosphorylation only at Ser³⁷⁵ and the morphine-induced phosphorylation is mediated by GRK5 (88–90). But there are marked differences in driving higher-order phosphorylation on flanking residues (Thr³⁷⁰, Thr³⁷⁶ and Thr³⁷⁹) between opioids. Multisite-phosphorylation induced by fentanyl, DAMGO, and etorphine requires GRK2/3 (88, 89). Also opioid induced receptor internalization is controlled by higher-order phosphorylation involving Thr³⁷⁰, Thr³⁷⁶ and Thr³⁷⁹ (89).

In case of the NOP receptor, it is known that GRK2 and GRK3 are relevant for NOP receptor desensitization (91–93). Additionally, GRK3 and ß-arrestin2 appear to be important for N/OFQ-induced internalization, because point mutation of Ser³⁶³ reduces, but not completely blocks, NOP receptor internalization (12). It is possible that several phosphorylation sites are essential for internalization and desensitization of the NOP receptor. Further, given the differential functional selectivity amongst chemically diverse reported NOP receptor agonists, it is possible that different agonists may induce recruitment of one or more GRK isoforms to the NOP receptor. Here, we investigated the mechanisms of agonist-induced NOP receptor phosphorylation by generating and extensively characterizing the first phosphosite-specific antibodies for the NOP receptor carboxyl-terminal residues Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³. These allowed us to selectively detect multiple phosphorylated forms of NOP receptor in vitro and in vivo after treatment with chemically diverse NOP receptor agonists.

Results

Phosphosite-specific antibodies reveal spatial and temporal dynamics of agonist-induced phosphorylation of Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ in the carboxyl-terminal tail of the NOP receptor.

The human NOP receptor contains a number of potential phosphate acceptor sites in its intracellular loops as well as within its carboxyl-terminal tail (Fig. 1A). In whole-cell phosphorylation assays, we observed a robust increase in NOP receptor phosphorylation after addition of its endogenous ligand N/OFQ (Fig. 1B). Notably, this increase was not observed in a NOP receptor mutant (6S/T-A) in which all six carboxyl-terminal serine and threonine residues were exchanged to alanine (Fig. 1B). This finding suggests that agonist-induced NOP receptor phosphorylation occurs primarily within its carboxyl-terminal tail. In an effort to examine the temporal and spatial dynamics of NOP receptor phosphorylation, we generated polyclonal phosphosite-specific antibodies for the carboxyl-terminal residues Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ (Fig. 1A). In addition, we also generated a polyclonal phosphorylation-independent antibody to the NOP receptor (NOPR) (Fig. 1A). First, antisera were affinity purified against their immunizing peptides and specificity was then verified with synthetic phosphopeptides and corresponding nonphosphopeptides using dot-blot assays (Fig. 1C). All antibodies, which clearly detected their respective peptide without cross-reaction with the nonphosphopeptide, were further characterized using Western blot analysis. The anti-pSer³⁴⁶ antibody, anti-pSer³⁵¹ antibody and anti-Thr³⁶²/Ser³⁶³ antibody specifically detected the respective Ser³⁴⁶-, Ser³⁵¹- or Thr³⁶²/Ser³⁶³-phosphorylated form of the NOP receptor after N/OFQ incubation of human embryonic kidney (HEK) 293 cells stably transfected with the human NOP receptor (Fig. 1, D and E). After treatment with lambda phosphatase, all phosphosite-specific antibodies were no longer able to detect their cognate forms of phosphorylated NOP receptors, whereas the receptor protein was still detectable using the phosphorylation-independent NOPR antibody (Fig. 1, D and E).

Figure 1: Characterization of phosphosite-specific NOP receptor antibodies.

(A) Schematic representation of the human nociceptin/orphanin FQ (hNOP) receptor. All potential intracellular phosphate acceptor sites are indicated (gray). Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ were targeted for the generation of phosphosite-specific antibodies and the epitope used for generating a phosphorylation-independent antibody (NOPR) is indicated by a black line. (B) HEK293 cells were transiently transfected with either the wild-type hNOP receptor or its 6S/T-A mutant. After 48 hours, cells were labeled with 200 μCi/ml carrier-free [³²P]orthophosphate. Labeled cells were either not treated (–) or treated (+) to 10 μM N/OFQ for 10 min, and whole-cell receptor phosphorylation was determined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. (C) Dot-blot analysis on serial dilutions of peptides P1-P4 to characterize antibody to pThr³⁶²/pSer³⁶³. (D and E) Characterization of phosphosite-specific antibodies directed against Ser³⁴⁶, Ser³⁵¹ or Thr³⁶²/Ser³⁶³ using λ-phosphatase. HEK293 cells stably expressing the HA-tagged hNOP receptor were either not treated (–) or treated (+) with 10 μM N/OFQ for 10 min. Lysates were then either incubated (+) or not (–) with λ-phosphatase and immunoblotted with the phosphosite-specific antibodies to pSer³⁴⁶, pSer³⁵¹, or pThr³⁶²/Ser³⁶³. Blots were stripped and reprobed with the phosphorylation-independent antibody to NOPR as a loading control. In all panels, blots are representative from one of three independent experiments. Molecular mass markers (kDA) are indicated, left.

For further characterization of the phosphosite-specific antibodies, different NOP receptor mutants were generated (Fig. 2A). Immunoblot analysis showed that N/OFQ strongly stimulated phosphorylation at Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³. As expected, receptor phosphorylation was not detectable after global mutation of all serine and threonine residues present in the carboxyl-terminal tail (6S/T-A) or in a mutant with additional mutation of all serine and threonine residues within the third intracellular loop (10S/T-A). No phosphorylation signal for pSer³⁴⁶ and pSer³⁵¹ was detectable in the S337A/S346A/S351A mutant (Fig. 2, B and C, left columns). Conversely, receptor constructs with a T362A/S363A/T365A mutation and an S363A point mutation displayed no binding of the anti-pThr³⁶²/pSer³⁶³ antibody after N/OFQ stimulation (Fig. 2B, middle column). It should be noted that the phosphorylation-independent NOPR antibody was not able to detect any protein band corresponding to the mutant construct T362A/S363A/T365A, suggesting that this sequence may contribute to the epitope recognized by the antibody (Fig. 2B, right column). Probing for the HA-epitope tag ensured that the T362A/S363A/T365A construct was indeed expressed, as well as all other NOP receptor constructs used in this study (Fig. 2, B and C, bottom row). These results confirm that we generated phosphosite-specific antibodies directed against Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ in the carboxyl-terminal tail.

Figure 2: Characterization of phosphosite-specific NOP receptor antibodies using receptor mutants.

(A) Sequence of the carboxyl-terminal tail of the hNOP receptor showing all potential phosphate acceptor sites. Serine (S) and threonine (T) residues depicted in gray were exchanged to alanine. (B and C) HEK293 cells stably expressing HA-tagged hNOP, S337/346/351A, T362/S363/T365A, S363A, 6S/T-A, or NOP-10S/T-A were either not treated (–) or treated (+) with 10 μM N/OFQ for 10 min, and lysates were then immunoblotted with the antibodies to pSer³⁴⁶, pSer³⁵¹, or pThr³⁶²/Ser³⁶³. Blots were stripped and reprobed with antibodies to NOPR or HA-tag. Blots are representative, (n=3).

NOP receptor phosphorylation occurs in a time-dependent manner with Ser³⁴⁶ as primary phosphorylation site followed by Ser³⁵¹ and Thr³⁶²/Ser³⁶³.

We then examined the time-course of N/OFQ-induced Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ phosphorylation and receptor internalization in vitro. After N/OFQ treatment, a robust phosphorylation at Ser³⁴⁶ and Ser³⁵¹ was detectable within 1 min, and remained abundant throughout the 60 min treatment period, whereas Thr³⁶²/Ser³⁶³ phosphorylation was first detectable after 1 min after the addition of N/OFQ and increased steadily throughout the 60 min treatment period (Fig. 3, A and B). To resolve the phosphorylation time-course in more detail, cells were exposed to N/OFQ at room temperature (RT) for shorter time periods. Under these conditions, S346 phosphorylation occurred within 20 s and Ser³⁵¹ phosphorylation within 60 s, whereas Thr³⁶²/Ser³⁶³ phosphorylation became first detectable after 3 min, suggesting that Ser³⁴⁶ is the primary site of phosphorylation, followed by Ser³⁵¹ and Thr³⁶²/Ser³⁶³ (Fig. 3, C and D).

Figure 3: Time-course of N/OFQ-induced Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ phosphorylation and internalization.

(A to D) HEK293 cells stably expressing hNOP receptors were exposed to 10 μM N/OFQ for the indicated times and temperatures [(A and B) up to 60 min at 37 °C; (C and D) up to 20 min at 22 °C (room temperature, RT)], and lysates were immunoblotted with the indicated antibodies. Blots are representative of n=5 (A and C) or 6 (B and D) independent experiments. Blots were stripped and reprobed for NOPR. (E) HEK293 cells stably expressing the HA-tagged hNOP receptor were preincubated with antibody to HA-tag and subsequently exposed to 10 μM N/OFQ for up to 60 min at 22 °C. Cells were fixed, permeabilized, immunofluorescently stained, and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 μm. (F) Stably HA-tagged hNOP receptor-expressing HEK293 cells were preincubated with antibody to HA-tag and stimulated with 10 μM N/OFQ for up to 90 min at 37 °C. Cells were then fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by enzyme-linked immunosorbent assay and quantified as the percentage of internalized receptors in agonist-treated cells. Data are means ± SEM of six independent experiments performed in quadruplicate.

The time-course of NOP receptor internalization was visualized by fluorescence microscopy and quantified using a cell-surface enzyme-linked immunosorbent assay (ELISA). NOP receptor internalization was first detectable after 5 min and reached a maximum after 60 min of agonist treatment (Fig. 3, E and F). These data indicate that N/OFQ-induced NOP receptor phosphorylation and internalization occur in a time-dependent manner with Ser³⁴⁶ as primary phosphorylation site followed by Ser³⁵¹ and Thr³⁶²/Ser³⁶³.

NOP receptor phosphorylation is mediated by GRK2 and GRK3.

Phosphorylation of GPCRs can be mediated by GPCR kinases (GRKs) or second messenger-activated kinases (such as PKA and PKC). We therefore incubated cells with phorbol 12-myristate 13-acetate (PMA) or forskolin and examined NOP receptor phosphorylation. Forskolin, which activates adenylyl cyclase (and consequently cAMP-dependent protein kinases), did not produce any detectable phosphorylation of Ser³⁴⁶, Ser³⁵¹ or Thr³⁶²/Ser³⁶³. However, activation of PKC by PMA induced selective phosphorylation of Ser³⁴⁶ and Ser³⁵¹, indicating that Ser³⁴⁶ and Ser³⁵¹ can also undergo heterologous PKC-mediated phosphorylation (Fig. 4A). To evaluate the contributions of GRKs to agonist-induced NOP receptor phosphorylation, we used chemical inhibitors as well as siRNA knockdown of GRK2/3. In fact, inhibition of GRK2/3 using the selective inhibitor compound 101 reduced N/OFQ-induced phosphorylation at Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ in a concentration-dependent manner (Fig 4, A and B). Treatment with specific GRK2 or GRK3 siRNA sequences also led to a significant reduction of N/OFQ-induced phosphorylation of these residues (Fig. 4C, fig. S1A). Given the close relationship between GRK2 and GRK3, it is possible that the loss of one isoform could be compensated for by the other isoform. Therefore, we evaluated the inhibitory effect of siRNA knockdown of both GRK2 and GRK3. Indeed, combined siRNA knockdown of both GRK isoforms produced a stronger inhibition of Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ phosphorylation, indicating that GRK2 and GRK3 function as a redundant system for agonist-induced NOP receptor phosphorylation (Fig. 4C, fig. S2A). To confirm these results, we performed GRK2 and GRK3 plasmid overexpression experiments. As expected, overexpression of GRK2 or GRK3 strongly enhanced N/OFQ-induced Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ phosphorylation (Fig. 4D, fig. S1B). These results suggest that GRK2 and GRK3 are responsible for N/OFQ-induced NOP receptor phosphorylation.

Figure 4: NOP receptor phosphorylation is mediated by GRK2 and GRK3.

(A) HEK293 cells stably expressing the hNOP receptor were preincubated with either vehicle (DMSO; control) or the GRK2/3-specific inhibitor compound 101 (cmpd 101) at 30 μM for 30 min at 37 °C, then exposed to 10 μM N/OFQ, 10 μM forskolin, or 1 μM PMA (or not, -) for 10 min. Lysates were then immunoblotted with antibodies to pSer³⁴⁶, pSer³⁵¹, or pThr³⁶²/Ser³⁶³. Blots were stripped and reprobed for NOPR. Blots are representative, n=3. (B) Cells described in (A) were preincubated with vehicle (DMSO; (–) or cmpd101 at the indicated concentrations for 30 min at 37 °C, then treated with water (–) or 10 μM N/OFQ for 10 min at 37 °C. Lysates were blotted as described in (A). Blots are representative, n=4. (C and D) Cells described in (A) were transfected with either siRNA targeting GRK2, GRK3, or GRK2 and GRK3 (GRK2/3) or a control (SCR) for 72 hours (C) or with GRK2 or GRK3 expression plasmids or an empty vector (MOCK) for 48 hours (D), then stimulated with 10 μM N/OFQ for 10 min at 37 °C. Lysates were immunoblotted with antibody to pThr³⁶²/Ser³⁶³. Blots were stripped and reprobed for NOPR. Densitometry, above the blots, was normalized to those in SCR- or MOCK-transfected cells, which were set to 100%. Data are mean ± SEM from five to six independent experiments. *P<0.05 vs. SCR or MOCK by two-way ANOVA with Bonferroni post-test.

Chemically diverse small-molecule NOP receptor agonists induce varying amounts of NOP receptor phosphorylation and internalization.

We next examined a range of chemically diverse NOP receptor ligands for their ability to induce NOP receptor phosphorylation and internalization. We consistently observed that the endocytotic activity of these agonists was associated with their ability to induce receptor phosphorylation at Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ (Fig. 5, A to D, and fig. S2). As expected, other endogenous or clinically-relevant opioids did not induce any detectable phosphorylation of these residues (fig. S3). Notably, our results reveal a correlation between G protein signaling and receptor phosphorylation, with NNC 63–0532 as an exception. Although MCOPPB, Ro64–6198, SCH221510, AT-202 and NNC 63–0532 strongly activated G protein signaling, only MCOPPB, Ro64–6198, SCH221510 and AT-202, but not NNC 63–0532, were able to induce robust multisite phosphorylation of the NOP receptor. Cebranopadol was also not capable of inducing either phosphorylation or NOP receptor internalization. To investigate the relationship between ligand-induced NOP receptor phosphorylation and internalization, phosphorylation values and internalization values were compared from every compound at a concentration of 10 μM (Fig. 5E and fig. S2). A direct positive linear correlation (r = 0.8581) was observed between the phosphorylation at Thr³⁶²/Ser³⁶³ and receptor internalization (Ser³⁴⁶: r = 0.8260 and Ser³⁵¹: r = 0.7877), indicating a relationship between the two events.

Figure 5: Agonist-selective NOP receptor phosphorylation and internalization.

(A) HEK293 cells stably expressing HA-tagged hNOP receptors were preincubated with HA antibody and then stimulated with 10 μM N/OFQ, Ro64–6198, MCOPPB, SCH221510, NNC 63–0532, AT-202, buprenorphine (BUP), norbuprenorphine (norBUP), cebranopadol or vehicle (according to the solvent) for 60 min at 22 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n=3 independent experiments. Scale bar, 20 μm. (B) NOP receptor-expressing HEK293 cells were treated with the compounds listed in (A) (–, vehicle solvent) at concentrations ranging from 10⁻⁹ to 10⁻⁵ M for 10 min at 37 °C, and lysates were immunoblotted with antibodies to pSer³⁴⁶, pSer³⁵¹, or pThr³⁶²/Ser³⁶³. Blots were stripped and reprobed for NOPR. Blots are representative of n=3–5 experiments. (C) HEK293 cells stably expressing hNOP receptors were preincubated with antibody to HA-tag and treated with vehicle (solvent) or 10 μM N/OFQ, Ro64–6198, MCOPPB, SCH221510, NNC 63–0532, AT-202, BUP, norBUP or cebranopadol for 60 min at 37 °C. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from twelve independent experiments performed in quadruplicate. (D) Maximum NOP receptor ligand-induced phosphorylation at Thr³⁶²/Ser³⁶³ from data in (B). Data are mean ± SEM from three independent experiments. *P<0.05 vs. N/OFQ by two-way ANOVA with Bonferroni post-test. (E) Correlation between NOP receptor phosphorylation and internalization induced by different ligands in HEK293 cells, from data in (A to D). Abscissae: ligand-induced internalization in percentage (normalized to N/OFQ). Ordinates: ligand-induced phosphorylation in percentage (normalized to N/OFQ). Solid line, linear regression of the data points; correlation coefficient r = 0.8581.

These results show that the NOP receptor phosphorylation and internalization is agonist-selective and may indicate biased coupling to intracellular signaling pathways in NOP receptors activated by NNC 63–0532.

Chemically diverse NOP receptor agonists show varying levels of functional efficacies in GIRK channel activation (G protein-dependent signaling).

To analyze G protein signaling of NOP receptor at high temporal resolution, we used a previously described fluorescence-based membrane potential assay that detects Gβγ-dependent activation of inwardly rectifying potassium (GIRK) channels (94). AtT-20 cells, a mouse pituitary tumor cell line, stably transfected with NOP receptor were loaded with membrane potential dye. Addition of N/OFQ induced a dose-dependent decrease in fluorescence intensity, which is indicative of membrane hyperpolarization (fig. S4, A and B). Half-maximal effective concentration (EC₅₀) for N/OFQ was 1.5 ± 0.4 nM. Overnight treatment with pertussis toxin (PTX) inhibited the N/OFQ-induced hyperpolarization (fig. S4C). Addition of SCH23390, a potent and selective GIRK channel blocker, reversed the N/OFQ-evoked hyperpolarization (fig. S4D). Collectively, these results indicate that NOP receptors can couple to GIRK channels endogenously expressed in AtT-20 cells. Next, we evaluated the ability of a variety of NOP receptor ligands to activate GIRK channels (Fig. 6, A to G). Notably, MCOPPB was the most potent agonist tested with an EC₅₀ of 0.06 ± 0.02 nM compared to N/OFQ (EC₅₀ of 1.5 ± 0.4 nM) (Fig. 6B). SCH221510 and Ro64–6198 exhibited similar dose-response curves with EC₅₀ values of 14.4 ± 3.2 nM and 15.9 ± 3.5 nM, respectively (Fig. 6, A and C). NNC 63–0532 and AT-202 (SR16835) are similar potent agonists with EC₅₀ values of 56.0 ± 9.3 nM and 29.2 ± 4.7 nM, respectively (Fig. 6, D and E). Buprenorphine and cebranopadol exhibited partial agonistic activity with a remarkably reduced maximal effect compared with N/OFQ (Fig. 6, F and G). Correlation analysis were done to investigate the relationship between ligand-induced NOP receptor phosphorylation and GIRK channel activation, phosphorylation values and E_max values from the GIRK channel activation assay were used from every ligand (Fig. 6H). A direct positive linear correlation with a correlation coefficient of r = 0.68659 was observed between the phosphorylation at Thr³⁶²/Ser³⁶³ and GIRK channel activation. These results show that NOP receptor mediated G protein signaling is agonist-dependent and that there exists a direct correlation between phosphorylation and GIRK channel activation.

Figure 6: G protein signaling of chemically diverse NOP receptor agonists.

(A to G) Agonist-induced hyperpolarization can be measured by changes in fluorescence intensity of fluorescent oxonol dyes. A reduction of fluorescent signal intensity is indicative of Gβγ protein-mediated GIRK channel activation. AtT20 cells stably expressing the hNOP receptor were first preloaded with the dye. Thereafter, a baseline is measured for 60 seconds before cells were stimulated with vehicle (according to the solvent) or with Ro64–6198 (A), MCOPPB (B), SCH221510 (C), NNC 63–0532 (D), AT-202 (E), BUP (F), cebranopadol (G) or N/OFQ (A to G) at a concentration range of 10⁻⁶ to 10⁻¹³ M for 180 seconds. Dose-response curves were calculated with OriginPro using sigmoidal non-linear fitting. Vehicle-induced changes in fluorescence signal (background) were subtracted from signals obtained using agonist-containing solutions. Data are mean ± SEM from three independent experiments performed in duplicate. ΔRFU is change in relative fluorescence. (H) Correlation between NOP receptor phosphorylation (pThr³⁶²/Ser³⁶³) and GIRK channel activation induced by the different ligands, color-coordinated with (A to G). Abscissae: ligand-induced G protein activation (EC₅₀ values). Ordinates: ligand-induced phosphorylation in percentage (normalized to N/OFQ). Solid line, linear regression of the data points; correlation coefficient r = 0.6859.

NOP receptor antagonists selectively inhibit N/OFQ-induced phosphorylation and internalization in vitro.

The opioid receptor antagonist naloxone did not inhibit NOP receptor phosphorylation or internalization after N/OFQ treatment (Fig. 7, A to D). In contrast, the selective NOP receptor antagonists J-113397 and SB 612111 (95, 96) completely blocked N/OFQ-induced phosphorylation at Ser³⁴⁶, Ser³⁵¹ and Thr³⁶²/Ser³⁶³ (Fig. 7, A and B). J-113397, but not naloxone, was also able to block N/OFQ-induced internalization (Fig. 7, C and D). Only selective NOP receptor antagonists could inhibit N/OFQ-induced phosphorylation and internalization.

Figure 7: Antagonist-selective inhibition of N/OFQ-induced phosphorylation, internalization and G protein signaling.

(A and B) HEK293 cells stably expressing hNOP receptors were preincubated (+) or not (–) with 50 μM naloxone, J-113397, or SB 612111 for 30 min at 37 °C, then treated with vehicle (water; -) or with 10 μM N/OFQ (+) for 10 min at 37 °C. Cell lysates were then immunoblotted with antibodies to pSer³⁴⁶, pSer³⁵¹, or pThr³⁶²/Ser³⁶³. Blots were stripped and reprobed for NOPR. Blots are representative, n=3. (C) HEK293 cells stably expressing the NOP receptor were preincubated with antibody to HA-tag and then treated with vehicle (DMSO), 50 μM naloxone, J-113397, or SB 612111 and with or without 10 μM N/OFQ for 60 min at 37 °C. Cells were then fixed and labeled with a peroxidase-conjugated secondary antibody, and receptor internalization was measured by ELISA and quantified as percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from six independent experiments performed in quadruplicate. *P<0.05 vs. N/OFQ by two-way ANOVA with Bonferroni post-test. (D) Cells described and treated as in (C), except treated at 22 °C, were fixed, permeabilized, immunofluorescently stained, and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 μm.

NOP receptor phosphorylation is also observed in vivo after systemic treatment with NOP receptor agonists.

Using the phosphosite-specific antibodies, we analyzed NOP receptor tissue distribution using Western blot. We were able to detect the NOP receptor in the brain, spinal cord and dorsal root ganglia (DRG), but not in the lung, heart, bladder, kidney and adrenal gland (Fig. 8A). For in vivo analysis of NOP receptor phosphorylation, the non-peptide full agonist AT-202 was administered by intraperitoneal (i.p.) injection to three mice. This NOP receptor agonist allows for systemic administration with significant central bioavailability (50, 54, 97). Several peptides from the third transmembrane helix and the C-terminal part of the mouse NOP receptor were identified by nanoLC-MS/MS under basal conditions and after in vivo AT-202 treatment. In particular, phosphorylation of Ser³⁴³ (equivalent to human Ser³⁴⁶) and Thr³⁵⁹/Ser³⁶⁰ (equivalent to human Thr³⁶²/Ser³⁶³) in the carboxyl-terminal tail (black symbols) could be demonstrated by CID/ETD fragmentation analysis (Fig. 8, B and C). The ³³⁹EMQVSDRVR³⁴⁷ peptide containing Ser³⁴³ was found only in its phosphorylated form in both saline and AT-202-treated samples (Fig. 8D). On the contrary, the ³⁵⁹TSETVPRPAGSIATMVSK³⁷⁶ peptide was detected in its Thr³⁵⁹ or Ser³⁶⁰ monophosphorylated form only in AT-202 samples (Fig. 8E) whereas the unphosphorylated peptide was present under both conditions. The lack of Thr³⁵⁹/Ser³⁶⁰ diphosphorylated peptide suggests that only phosphorylation of either Thr³⁵⁹ or Ser³⁶⁰ is induced by AT-202. These data indicate that NOP receptor is expressed in the mouse central nervous system and that Ser³⁴³ and Thr³⁵⁹/Ser³⁶⁰ are important phosphorylation sites in vivo after agonist treatment.

Figure 8: Tissue distribution of NOP receptor and NanoLC-MS/MS analysis of NOP receptor phosphorylation in vivo.

(A) Distribution of NOP receptor in NOP-eGFP knock-in mouse tissue. Anesthetized NOP-eGFP knock-in mice were sacrificed and tissues were removed. The NOP receptor was then immunoprecipitated from homogenates with anti-GFP protein agarose beads, and samples were immunoblotted with the phosphorylation-independent antibody to NOPR or antibody to GFP. Bottom, prolonged ECL detection exposure. Blots are representative from one of three independent experiments. (B) Mass spectrometry coverage of the NOP receptor sequence from mouse brain. The schema represents the secondary structure of the mouse NOP receptor. Filled (blue and black) symbols indicate the protein sequence covered by nanoLC-MS/MS; the phosphorylated residues identified are black. Red circles indicate the trypsin cleavage sites. Pm, plasma membrane; e1-e3, extracellular loops; i1-i4, intracellular loops (C) List of phosphorylated and unphosphorylated NOP receptor peptides identified by nanoLC-MS/MS in the mouse brain. Amino acids belonging to the GFP sequence are in italics. Theo. Mass., theoretical mass (DA); MC, missed cleavage. (D and E) ETD MS/MS spectra of the monophosphorylated peptide 339-EMQVpSDRVR-347 (D; triply charged precursor ion, MH3+, at m/z 400.5128) and 359-pT/pSETVPRPAGSIATMVSK-376 (E; triply charged precursor ion, MH3+, at m/z 637.9797) display series of c- and z-ions indicating that Ser³⁴³ (D) and Thr³⁵⁹ or Ser³⁶⁰ (E) are phosphorylated, respectively. Red labels indicate site-determining ions and the corresponding peaks in the spectrum. Blue labels indicate fragment ions that confirm the site localization and exclude another potential site. pS, pT: phosphorylated serine or threonine residues.

Agonist-selective NOP receptor phosphorylation and internalization in mouse brain.

To analyze in more detail the agonist-induced NOP receptor phosphorylation in vivo, NOP-eGFP mice were treated with different types of NOP receptor ligands. The corresponding mouse NOP receptor phosphorylation sites are located at equivalent positions compared to the human NOP receptor (Fig 9A). This fact enables us to use the phosphorylation-dependent antibodies directed against human residues to detect agonist-induced phosphorylation at mouse residues Ser³⁴³ (equivalent in human Ser³⁴⁶), Ser³⁴⁸ (equivalent in human Ser³⁵¹) and Thr³⁵⁹/Ser³⁶⁰ (equivalent in human Thr³⁶²/Ser³⁶³). To analyze NOP receptor internalization in vivo, ventral midbrain neurons from NOPR-eYFP mice were used. The MS data as well as the in vivo Western blot phosphorylation data show that there is a constitutive phosphorylation of Ser³⁴⁶ and Ser³⁵¹ in mouse brain. The AT-202-induced phosphorylation signal occurs in a concentration-dependent manner (Fig. 9B, fig. S5A) and could be blocked by the NOP receptor-selective antagonist SB 612111 (Fig. 9C, fig. S5B). To evaluate the in vivo contribution of GRKs to AT-202-induced NOP receptor phosphorylation, we used the selective GRK2/3 inhibitor compound 101. In fact, inhibition of GRK2/3 using compound 101 blocked the AT-202-induced phosphorylation at Thr³⁶²/Ser³⁶³ in a concentration-dependent manner (Fig. 9D). These results suggest that GRK2 and GRK3 are also responsible for AT-202-induced NOP receptor phosphorylation in vivo. AT-202, Ro64–6198 and SCH221510, but not NNC 63–0532, induced strong NOP receptor phosphorylation at Ser³⁴³ (equivalent in human Ser³⁴⁶), Ser³⁴⁸ (equivalent in human Ser³⁵¹) and Thr³⁵⁹/Ser³⁶⁰ (equivalent in human Thr³⁶²/Ser³⁶³), as well as receptor internalization (Fig. 9, E to G, fig. S5C). Only a weak escalation of phosphorylation signal was detectable after administration of NOP receptor agonist MCOPPB in comparison to the treatment with saline at Ser³⁴⁸ (equivalent in human S351) and Thr³⁵⁹/Ser³⁶⁰ (equivalent in human Thr³⁶²/Ser³⁶³) (Fig. 9E, fig. S5C). In contrast, there was a strong MCOPPB-induced NOP receptor internalization and phosphorylation signal at Ser³⁴³ (equivalent in human Ser³⁴⁶) detectable (Fig. 9, E to G, fig. S5C). Moreover, addition of NOP receptor antagonist J-113397 blocked the N/OFQ-induced internalization (Fig. 9, F and G). These results indicate that the NOP receptor phosphorylation and internalization occurs in an agonist-selective manner.

Figure 9: Agonist-selective NOP receptor phosphorylation in mouse brain.

(A)Schematic representation of the human (h) and mouse (m) nociceptin/orphanin FQ receptor. All potential intracellular phosphate acceptor sites are indicated (gray). (B) After i.p. injection of 0.9% NaCl (0) or AT-202 (0.3 to 30 mg/kg) for 30 min, NOP-eGFP knock-in mice were euthanized and brains were removed. NOP receptor was immunoprecipitated with anti-GFP protein agarose beads and immunoblotted with antibodies to pSer³⁴⁶, pSer³⁵¹, or pThr³⁶²/Ser³⁶³. Blots were stripped and reprobed for NOPR or GFP. Blots are representative, n=3. (C) As in (B), but mice were treated with SB 612111 or AT-202 singly or pretreated with SB 612111 for 30 min followed by AT-202 (each 30 mg/kg). (D) After intracerebroventricular injection of compound 101 (0.3 to 30 nmol), NOP-eGFP knock-in mice were treated with AT-202 (30 mg/kg for 30 min), euthanized and brains were removed. Homogenates underwent immunoprecipitation with anti-GFP agarose beads, and the resulting samples were immunoblotted for phosphorylated (pThr³⁶²/Ser³⁶³) NOP receptor. Blots were stripped and reprobed with the GFP antibody as a loading control. Blots are representative, n=3. (E) As in (B), but mice were treated with 0.9% NaCl (–) or 30 mg/kg AT-202, Ro64–6198, NNC 63–0532, SCH221510 or MCOPPB for 30 min. (F and G) Imaging (F) and analysis (G) of NOP receptor internalization in ventral midbrain neurons. Primary cultures from NOPR-eYFP mice were treated for 1 hour with 1 μM of the indicated agonist, followed by live cell spinning disk confocal imaging. Images are representative, and data are means ± SEM of over 40 images from at least two dishes per condition.

Discussion

Using the first phosphosite-specific antibodies for the NOP receptor, we found evidence for hierarchical and temporally controlled multisite-phosphorylation of NOP receptors, both in vitro and in vivo. Moreover, we detected distinctive ligand-selective phosphorylation patterns. The canonical model for GPCR activation/deactivation cycle postulates the following steps: The agonist-bound receptor/G protein complex becomes a substrate for GRKs or second messenger-dependent protein kinases. Phosphorylation by GRKs increases the affinity for β-arrestins, which uncouple the receptor from its G protein and target it to clathrin-coated pits for internalization, thus desensitizing the primary signaling while simultaneously initiating β-arrestin-dependent signaling. Internalized GPCRs are either trafficked to lysosomes for degradation or recycle back through an endosomal pathway.

Agonist-induced phosphorylation has been studied in great detail for several GPCRs, most notably β2-adrenoceptor (β2-AR), MOP receptor, somatostatin sst2, sst3 and sst5 receptor subtypes, and angiotensin II AT1a receptors (87–89, 98–110). The emerging picture obtained from these studies suggests that the endogenous agonist N/OFQ induces phosphorylation that proceeds in a temporal hierarchy, involving multiple serine and threonine residues located in the carboxyl-terminal domain of the NOP receptor. In each GPCR case, a primary phosphate-acceptor site has been identified and phosphorylation levels positively correlate with receptor internalization levels. In contrast, many synthetic agonists were found to produce only partial receptor phosphorylation, combined with reduced agonist-stimulated internalization. These observations support the general hypothesis that exhaustive GPCR phosphorylation is a prerequisite for internalization and a hallmark of full agonists.

For human NOP receptors, we identified Ser³⁴⁶ as the primary phosphorylation site and hierarchical phosphorylation continued thereafter to include Ser³⁵¹, Thr³⁶² and Ser³⁶³. Stimulation with N/OFQ induced maximal receptor internalization, although with slower kinetics than the closely related MOP receptor (88, 106). Thr³⁶²/Ser³⁶³ in the NOP receptor are phosphorylated by GRK2/3-mediated phosphorylation in vitro. In contrast, Ser³⁴⁶ and Ser³⁵¹ are also substrates for heterologous PKC phosphorylation. In MOP receptor also two phosphorylation sites (Thr³⁷⁰ and Ser³⁶³) are phosphorylated by PKC (106). In vivo, AT-202-induced Thr³⁶²/Ser³⁶³ phosphorylation is also predominantly mediated by GRK2/3. Nevertheless, a function of GRK5/6 in NOP receptor regulation and signaling cannot be ruled out.

Similar to MOP receptor agonists (88–90), most synthetic NOP receptor agonists displayed reduced potencies to stimulate receptor phosphorylation, which appeared to correlate with the level of agonist-induced internalization. One notable exception is MCOPPB, which has been described as 10-fold more potent than N/OFQ in inhibiting cAMP accumulation and displaying strong G protein-biased signaling (78, 111). Our results from GIRK channel activation assay support these previous findings. MCOPPB was also the only synthetic agonist that exhibited similar potency as N/OFQ to induce NOP receptor phosphorylation in vitro. NOP receptor agonists SCH221510 and Ro64–6198 have been previously described as full agonists in G protein-dependent cAMP assay (55, 78). We confirmed these results with our study. Agonist AT-202 was described as a modestly selective NOP receptor full agonist, with very low efficacy at MOP receptor (54). In our GIRK channel activation assay, we confirmed that AT-202 acted as a NOP receptor full agonist. In contrast, the small-molecule NOP receptor agonist NNC 63–0532 failed to induce any phosphorylation at the NOP receptor, but behaved as a full agonist in G protein-mediated GIRK channel activation, albeit with about 40-fold reduced potency compared to N/OFQ. The opioid agonist buprenorphine, which also shows low agonist efficacy at NOP receptor (67, 112–116), displayed partial agonism in G protein-mediated GIRK assay. NNC 63–0532 and buprenorphine had previously been described as partial agonists at the NOP receptor in cAMP inhibition assays (78). Notably, the same study revealed an almost complete absence of β-arrestin recruitment, which is consistent with the complete lack of NOP receptor phosphorylation by both agonists. Morphine and fentanyl are the most effective drugs for the treatment of severe pain. But severe side effects, misuse of opioids and opioid addiction limit their use in clinical setting so that the development of addiction-free effective opioids for treating severe pain is required. Cebranopadol is the first bifunctional MOP/NOP receptor agonist that is in advanced clinical trials for the treatment of acute and chronic pain (58, 59) and has previously been described as full agonist in calcium mobilization and BRET studies (63) as well as in GTPγS assays (59). In our phosphorylation studies as well as the GIRK channel activation assay, cebranopadol clearly behaved as partial agonist at the NOP receptor. AT-121 is another bifunctional partial agonist at MOP/NOP receptor that is able to suppress oxycodone-induced reinforcing effects and elicit morphine-like analgesic effect without common side effects of classical opiods (66). Full NOP receptor agonists such as Ro64–6198 or AT-202 often induce a strong decrease in locomotor activity in vivo which limits their clinical utility (50, 117). Both cebranopadol and AT-121 are devoid of this activity suggesting that partial agonism may be a desired property for the development of bifunctional MOP/NOP receptor ligands as effective painkillers with fewer side effects.

Localization of NOP receptor has been characterized using in vitro autoradiography, in situ hybridization and immunofluorescence staining (1–3, 22–26, 118–121). Most notably, we could detect NOP receptor in the brain, spinal cord and dorsal root ganglia. from untreated mice by mass spectrometry and Western blotting. Studies have used the same techniques to analyze phosphorylation at MOP receptors (88, 103, 106, 122). In case of NOP receptor, Ser³⁴⁶, Thr³⁵⁹ and Ser³⁶⁰, were uncovered in the brain after AT-202 administration, whereas Ser³⁴³ and Ser³⁴⁸ are constitutively phosphorylated in the absence of agonist. This basal phosphorylation may reflect high constitutive activity of the NOP receptor. Nevertheless, after agonist injection, an increase of phosphorylation at both these sites was also observed. AT-202-induced phosphorylation occurred in a dose-dependent manner and could be blocked with SB 612111. In accordance with the corresponding human phosphorylation sites, in vivo phosphorylation in mice occurred in an agonist-selective manner, similarly to the MOP receptor (103). MCOPPB administration enhanced NOP receptor phosphorylation only at Ser³⁴³ and Thr³⁵⁹/Ser³⁶⁰ but not Ser³⁴⁸. In MOP receptor, in vivo, morphine induced phosphorylation only at Ser³⁷⁵ (103). NOP receptor internalization in vivo occurred also in an agonist-dependent manner and could be blocked by J-113397.

In conclusion, we identified an agonist-selective phosphorylation pattern in the carboxyl-terminal domain of the NOP receptor in vitro and in vivo that correlates with receptor internalization. It is conceivable that the selective phosphorylation patterns provide an indication of a general biochemical mechanism by which the different functional effects of N/OFQ might be explained. Further, differential phosphorylation patterns amongst non-peptidic NOP receptor agonists suggests that structure-function correlations may be possible for the NOP receptor and may explain the differences in NOP receptor agonist pharmacology. This study provides important tools to characterize agonist-dependent regulation of NOP receptor signaling at the cellular level, which may be beneficial for the development and characterization of NOP receptor agonists for therapeutic indications.

Materials and Methods

Animals

NOP-eGFP mice (8–12 weeks old) were used to detect NOP receptor phosphorylation in vivo. Animals were group-housed under standard laboratory conditions and kept on a 12 hours day/night cycle (lights on at 7:00 A.M.) at constant temperature (20–22 °C) and humidity (45–55 °C) with ad libitum access to food and water. Animals were handled three times before the experiment. Mice were maintained in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the Thuringian state authorities and complied with European Commission regulations for the care and use of laboratory animals. All methods used were preapproved by the Institutional Animal Care and Use Committee at the Torrey Pines Institute for Molecular Studies (Port St Lucie) and the University Hospital Jena, Institute of Pharmacology and Toxicology (Jena).

NOPR-eYFP mice were used to detect NOP receptor internalization in primary neurons. For these mice, eYFP was knocked into exon 4 of NOP receptor and fused at the C-terminus of the receptor, flanked on both sides by loxP sites. Embryonic mice were removed from euthanized pregnant female mice on gestational day 14 (E14) and used for ventral midbrain dissection. All procedures were approved by the Animal Care and Use Committee of Washington University and adhered to NIH guidelines.

Plasmids

DNA for human NOP receptor and human NOP receptor mutants were generated via artificial synthesis and cloned into pcDNA3.1 by imaGenes and Eurofins, respectively. The coding sequence for an amino-terminal HA-tag was added. Human GRK2 and GRK3 plasmids were obtained from OriGene and TransOMIC, respectively.

Antibodies

Peptide sequences used for generating phosphosite-specific antibodies against individual phosphorylated forms of the NOP receptor are shown in Table 1, including a phosphorylation-independent antiserum targeting a distal epitope in the NOP receptor carboxyl-terminal domain. The respective peptides were coupled to keyhole limpet haemocyanin after HPLC purification. The conjugates were mixed 1:1 with Freundś adjuvant and injected into groups of two or three rabbits (5033, 5034) for anti-pSer³⁴⁶ antibody production, (4876, 4878) for anti-pSer³⁵¹ antibody production, (4873–4875) for anti-pThr³⁶²/Ser³⁶³ antibody production, and (4870–4872) for anti-NOP receptor antibody production. The rabbits were injected at 4-week intervals and serum was obtained 2 weeks after immunizations, beginning with the second injection. Using dot blot analysis, specificity of the antisera was tested. For subsequent analysis, antibodies were affinity-purified against their immunizing peptide, immobilized using the SulfoLink kit (Thermo Scientific). Generation and characterization of the polyclonal rabbit anti-HA antibody was previously described (123). Anti-GRK2 (sc-562), anti-GRK3 (sc-563) and anti-rabbit IgG HRP-coupled (sc-2004) antibodies were obtained from Santa Cruz Biotechnology. The anti-HA IgG CF™488A antibody (SAB4600054) was purchased from Sigma-Aldrich. Anti-rabbit Alexa488-coupled antibody (A11008) was obtained from Invitrogen.

Table 1: NOP receptor peptide sequences used for generation of phosphosite-specific antisera.

List of peptide sequences used for generating phosphosite-specific antibodies against individual phosphorylated forms of the NOP receptor and a phosphorylation-independent antiserum targeting the NOP receptor at the end of the carboxyl-terminal domain. Endogenous cysteine were exchanged (abu).

Antiserum Name	Sequence used for immunization	Amino acid position in human NOP receptor
Ser346	REMQV-(p)S-DRVR	341–350
Ser351	DRVR-(p)S-IAKDV	347–356
Thr362/Ser363	LG-abu-K-(p)T- (p)S-ETVPR	358–368
NOPR (phosphorylation-independent)	VRSIAKDVGLG-abu-KTSETVPRPA	349–370

Drugs

The NOP receptor agonist AT-202 was synthesized and provided by Astraea Therapeutics. Ro64–6198 was provided by Roche. Nociceptin (ab120070) and dynorphin A (ab120412) were obtained from Abcam. MCOPPB (PZ0159), ß-endorphin (E6261), DADLE (E7131), DAMGO (E7384), etonitazene (E5007), etorphine, fentanyl (F3886), hydromorphone (H5136), [Met]-enkephalin (M6638), morphine-6-glucuronide (M3528), oxycodone (O1378), pentazocine (P134), tapentadol (T058), naloxone (N7758) and PMA (P8139) were purchased from Sigma-Aldrich. SCH221510 (3240), NNC 63–0532 (1780), J-113397 (2598), SB 612111 (3573), DPDPE (1431), endomorphin-1 (1055), endomorphin-2 (1056), U50488 (0495), PTX (3097), SCH23390 (0925) and forskolin (1099) were obtained from Tocris. Norbuprenorphine (BUP-982-FB) and buprenorphine (BUP-399-HC) were purchased from Lipomed. γ-Endorphin (60893–02-9) was obtained from Bachem and tramadol from Grunenthal. Levomethadone (00424906) and pethidine (03012446) were purchased from Sanofi-Aventis. Morphine (26–6) was obtained from Merck Pharma. Nalfurafine (A12579) was purchased from Adooqioscience LLC. Piritramid and sufentanil were obtained from Hameln Pharma Plus. Remifentanil was purchased from GlaxoSmithKline. Nortilidine was obtained from Pfizer AG. Cebranopadol (HY-15536) was purchased from MedChem Express. Lambda-phosphatase (P0753S) was obtained from Santa Cruz. Compound 101 (HB2840) was obtained from Hello Bio. Tertiapin-Q was purchased from Alomone Labs. AT-202, Ro64–6198, PMA, SCH221510, NNC 63–0532, J-11339, SB 612111, DPDPE, U50488, forskolin, nalfurafine and cebranopadol are DMSO-soluble and all the other mentioned compounds are water-soluble.

Cell culture and transfection

Human embryonic kidney 293 (HEK293) cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Deutsche Sammlung von Mikroorganismen und Zellkulturen; DSMZ). AtT20-D16v-F2 (AtT20) cells were purchased from American Type Tissue Culture Collection. All cells were cultured at 37 °C and 5% CO₂ in Dulbeccós modified Eaglés medium (DMEM), supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin. HEK293 cells and AtT20 cells were stably transfected with TurboFect (ThermoFisher Scientific). Stable transfected cells were selected in medium supplemented with 400 μg/ml geneticin.

Fluorescence-activated cell sorting (FACS)

To increase the number of HEK293 cells or AtT20 cells stably expressing the hNOP receptor or mutant hNOP receptors, FACS was used. Cells were incubated with trypsin, washed with PBS, resuspended in OPTI-MEM containing anti-HA IgG, CF™488 antibody (Sigma-Aldrich) and incubated for 30 min at room temperature. Again, cells were centrifuged and resuspended in FACS buffer (1 mM EDTA; 25 mM HEPES, pH 7.0; 1% BSA in PBS without Ca²⁺ and Mg²⁺). Subsequently, cells were strained (strainer 40 μm). Transfected cells were sorted using a FACS Aria III cell sorter (BD Biosience; 488 nm argon laser) and thereafter recultivated as described. This process was repeated 2–3 times to increase enrichment of stably transfected cells.

Small interfering RNA (siRNA) silencing of gene expression

Chemically synthesized double-stranded siRNA duplexes (with 3′-dTdT overhangs) were obtained from Qiagen for the following targets: GRK2 (5′-AAGAAAUUCAUUGAGAGCGAU-3′) and GRK3 (5′-AAGCAAGCUGUAGAACACGUA-3′), and from GE Dharmacon a non-silencing RNA duplex (5′-GCUUAGGAGCAUUAGUAAA-3′ and 3′-UUUACUAAUGCUCCUAAGC-5′). Stably HA-hNOP receptor expressing HEK293 cells were transfected with 150 nM siRNA for single transfection or with 100 nM of each siRNA for double transfection for 3 days using HiPerFect. All experiments showed target protein abundance reduced by ≥80%.

Whole cell phosphorylation assays

Phosphorylation studies were carried out as described (123–125). Stable HA-hNOP receptor or 6S/TA expressing HEK293 cells were labeled with [³²P]orthophosphate (285 Ci/mg P_i; ICN, Eschwege) for 60 min at 37 °C. Labeled cells were incubated with 10 μM N/OFQ or vehicle for 10 min at 37 °C. Subsequently cells were placed on ice and scratched into precipitation buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 10 mM NaF; 10 mM disodium pyrophosphate; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS) in the presence of protease and phosphatase inhibitors cOmplete mini® and PhosSTOP® (Roche Diagnostics) and solubilized. HA-tagged hNOP receptors were immunoprecipitated using 50 μl anti-HA-agarose beads (ThermoFisher Scientific) and receptors were eluted in SDS-sample buffer for 30 min at 50 °C. Samples were size-separated on 7.5% SDS polyacrylamide gels followed by autoradiography.

Western blot analysis

Stably transfected cells were plated onto poly-L-lysine-coated 60-mm dishes and grown for 2 days to 80% confluency. After treatment with agonists or antagonists, cells were lysed with detergent buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 10 mM NaF; 10 mM disodium pyrophosphate; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS) in the presence of protease and phosphatase inhibitors. Where indicated, cells were preincubated with GRK2/3 inhibitor compound 101 or NOP receptor antagonists for 30 min before agonist exposure. After 30 min centrifugation at 4 °C, HA-tagged hNOP receptors were enriched using anti-HA-agarose beads. Samples were inverted for 1.5 hours at 4 °C. Where indicated, samples were dephosphorylated with lambda protein phosphatase (Santa Cruz) for 1 hour at 30 °C. After washing, proteins were eluted using SDS sample buffer for 30 min at 50 °C. Proteins were separated on 7.5% or 12% SDS-polyacrylamide gels, and after electroblotting, membranes were incubated with 0.1 μg/ml antibodies to pSer³⁴⁶ (5034), pSer³⁵¹ (4876) or pThr³⁶²/Ser³⁶³ (4874) overnight at 4 °C, followed by detection using enhanced chemiluminescence detection (ECL) of bound antibodies (Thermo Fisher Scientific). Blots were subsequently stripped and reprobed with the phosphorylation-independent antibody to NOPR (4871) or antibody to HA-tag (0631) to ensure equal loading of the gels.

In vivo phosphorylation studies

First, the distribution of NOP receptor in NOP-eGFP mouse brain and periphery was examined. Briefly, mice (n=3) were euthanized under deep anesthesia and organs (brains, spinal cord, dorsal root ganglia, lungs, heart, kidneys, adrenal glands) were collected, flash frozen on dry ice and stored at −80 °C until biochemical analysis. In a second set of experiments, the ability of different NOP receptor agonists to induce NOP receptor phosphorylation in vivo was investigated. Mice (n=3 per compound) were given i.p. injections of NOP receptor agonists MCOPPB (30 mg/kg), SCH221510 (30 mg/kg), NNC 63–0532 (30 mg/kg), AT-202 (30 mg/kg) or Ro64–6198 (30 mg/kg), or NOP receptor antagonist SB 612111 (30 mg/kg), or vehicle. Animals were sacrificed 30 min after treatment. Brains were immediately dissected out, frozen on dry ice and stored at −80 °C until biochemical analysis. A dose-response study for NOP receptor agonist AT-202 to induce NOP receptor phosphorylation was also performed using NOP-eGFP animals. Mice (n=3 per dose) were given i.p. injections of different doses of AT-202 (0, 0.3, 3, 10, 30 mg/kg). Samples were weighed, transferred into ice-cold detergent buffer (50 mM Tris-HCL, pH 7.4; 150 mM NaCl; 5 mM EDTA; 10 mM NaF; 10 mM disodium pyrophosphate; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS; containing protease and phosphatase inhibitors) and homogenized using MINILYS workplace homogenizer (Peqlab). After 1 hour lysis at 4 °C, tissue homogenates were centrifuged at 16000 × g for 30 min at 4 °C. Supernatants were immunoprecipitated with anti-GFP antibody (Roche) bound to protein G-agarose beads or anti-GFP beads (NanoTag) for 1.5 hours at 4 °C. After protein determination using a Bradford Assay Kit (Thermo Fisher Scientific), proteins were eluted from the beads with SDS-sample buffer for 30 min at 50 °C. Proteins were separated on 7.5% SDS-polyacrylamide gels and after electroblotting membranes were incubated with antibodies to pSer³⁵¹ (4878), pSer³⁴⁶ (5034) or pThr³⁶²/Ser³⁶³ (4874) at a concentration of 0.1 μg/ml, followed by ECL detection of bound antibodies. Blots were stripped and reprobed with the phosphorylation-independent antibody to NOPR (4871) or antibody to GFP (Synaptic Systems) to confirm equal loading of the gels.

Analysis of NOP receptor internalization

Stably transfected HA-tagged hNOP receptor cells or receptor mutant cells were plated onto poly-L-lysine-coated coverslips and grown overnight. Cells were incubated with rabbit anti-HA antibody (0631) in serum-free medium for 2 hours at 4 °C. After agonist or antagonist exposure for 60 min at room temperature, cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 30 min at room temperature. Subsequently, cells were washed several times with phosphate buffer (22.6 ml/L 1 M NaH₂PO₄•H₂O; 77.4 ml/L 1 M Na₂HPO₄•H₂O; 0.1 % Triton X-100, pH 7.4), permeabilized and then incubated with an Alexa488-coupled goat anti-rabbit antibody (Invitrogen). Cells were mounted and internalization was examined using a Zeiss LSM510 META laser scanning confocal microscope. For quantitative internalization assays, cells were plated onto 24-well plates and grown overnight. Cells were preincubated with anti-HA antibody (0631) for 2 hours at 4 °C and then exposed to agonists or antagonists for 60 min at 37 °C. Subsequently, cells were fixed for 45 min at room temperature, washed 3 times with PBS and incubated with a peroxidase-conjugated secondary antibody (Santa Cruz). After washing, ABTS substrate was added and optical density was measured at 405 nm using a FlexStation 3 microplate reader (Molecular Devices).

Primary culture of ventral midbrain neurons.

Ventral midbrain neurons were cultured using a procedure adapted from (126). The ventral mesencephalon was removed from embryonic day 14 (E14) NOPR-eYFP mice. Tissues were mechanically dissociated, incubated with 0.25% trypsin and 0.05% DNase in DPBS for 20 min at 37 °C, and triturated using ART 200 REACH Barrier pipette tips (ThermoFischer). Glass bottom dishes (Cellvis) were pre-coated overnight with 2 mg/ml poly-D-lysine, then rinsed three times with sterile water and dried prior to use on the day of dissections. Trituration and initial plating was performed in DMEM+ (DMEM/F12 with HEPES, supplemented with 10% fetal bovine serum, 1x B27 additive, 1 μg/ml glucose, and 1x penicillin-streptomyocin). Dissociated neurons were diluted to 1 million cells/ml in DMEM+ and plated as a 50 μl drop in the center well of the glass bottom dishes. The dishes were incubated for 1 hour at 37 °C, 5% CO₂ to let the cells adhere, then 2ml of a 2:1 solution of DMEM+:NB+ was added to the dish (NB+: neurobasal medium supplemented with B27 additive, 1x penicillin/streptomycin, and L-glutamine). Every 2–3 days, one half of the medium was replaced with NB+.

NOP receptor internalization studies in primary ventral midbrain neurons.

Dissociated cultures of ventral midbrain neurons from NOPR-eYFP mice were imaged at DIV 7–10 using an Andor Revolution imaging system consisting of a Leica DMI6000B microscope, a Yokogawa CSU-X1 spinning-disk unit, an Andor iXon electron-multiplying charge-coupled device camera, and a laser combiner with four solid state lasers, all controlled using Andor iQ3 software. All imaging was performed inside a temperature-controlled chamber held at 37 °C, 5% CO₂. Images were acquired using a 63x oil immersion objective, 515 nm laser excitation, and 8 sec exposure time.

Membrane potential assay

Membrane potential was measured as previously described (94). Stably HA-hNOP receptor transfected AtT20 cells were plated into 96-well plates. Cells were washed with Hankś balanced salt solution (HBSS), buffered with 20 mM HEPES pH 7.4, containing 1.3 mM CaCl₂; 5.4 mM KCl; 0.4 mM K₂HPO₄; 0.5 mM MgCl₂; 0.4 mM MgSO₄; 136.9 mM NaCl; 0.3 mM Na₂HPO₄; 4.2 mM NaHCO₃; 5.5 mM glucose. Subsequently, cells were incubated with membrane potential dye (FLIPR Membrane Potential kit BLUE, Molecular Devices) for 45 min at 37 °C. Compounds and vehicle were injected in a final volume of 20 μl. The initial volume in the wells was 180 μL (90 μL buffer plus 90 μL dye) and 20 μL of compound was added to the cells resulting in a final volume in the well of 200 μL and a 1:10 dilution of the compound. Hence, the compounds were prepared at 10x concentrations. A baseline reading is measured for 60 seconds before compounds or buffer were injected. Measurements were accomplished at 37 °C using a FlexStation 3 microplate reader (Molecular Devices). The data was first normalized to the baseline and then subtracted from the buffer-only trace for each corresponding data point.

NanoLC-MS/MS analysis

After i.p. administration of saline or AT-202 (30 mg/kg) and GFP immunoprecipitation from 3 brains per condition, immunoprecipitates were boiled in SDS sample buffer for 5 min at 100 °C. Subsequently, brain extracts were alkylated in 90 mM iodoacetamide for 30 min in the dark, separated by SDS-PAGE on 10% polyacrylamide gels followed by gel staining with colloidal Coomassie blue. At the molecular weight of NOP receptor a band was excised and subjected to in-gel tryptic digestion using modified porcine trypsin (Promega) at 20 ng/μl. The dried peptides were extracted and analyzed by on-line nanoLC using an Ultimate 3000 system (Dionex) coupled to an ETD-enabled LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) as described (122, 127). The survey scan MS was performed in the Orbitrap on the 300–2000 m/z mass range with the resolution set to a value of 60.000 at m/z 400. The 20 most intense ions per survey scan were selected for subsequent CID/ETD fragmentation, and the resulting fragments were analyzed in the linear trap (LTQ). The settings for the data-dependent decision tree-based CID/ETD method were as follows: ETD was performed for all precursor ions with charge states > 5. The normalized collision energy was set to 35% for CID. The reaction time was set to 100 ms and supplemental activation was enabled for ETD. Triple technical replicates were performed in some conditions.

All raw mass spectrometry files were processed with MaxQuant (v 1.5.5.1) for database search with the Andromeda search engine. Data were searched against SwissProt database with taxonomy Mus musculus (16761 sequences) implemented with the mouse NOP receptor-eGFP sequence. Enzyme specificity was set to trypsin/P and a maximum of three missed cleavages were allowed. Carbamidomethylation of cysteine was set as fixed modification whereas oxidation of methionine and phosphorylation of serine, threonine and tyrosine were set as variable modifications. The precursor mass tolerance was set to 20 ppm for the first search and 10 ppm for the main Andromeda database search. The mass tolerance in MS/MS mode was set to 0.8 Da. The required minimum peptide length was seven amino acids, and minimum number of unique peptides was set to one. Andromeda results were validated by the target-decoy approach using a reverse database and the false discovery rates (FDR) at the peptide-spectrum matches (PSM), protein and site levels were set to 1%. Phosphosite localization was evaluated based on the Phosphosite Localization Scoring and Localization Probability algorithm of the Andromeda search engine.

Data Analysis

Protein bands detected on Western blots were quantified using ImageJ 1.47v software. Data were analyzed using GraphPad Prism 5 software. Densitometry of every protein band was carried out with Image J. We used the same area size to perform densitometry for every protein band from the same experiment for every phosphorylation site as well as the total receptor. Accordingly, an equally sized, empty area from the blot/film was measured to subtract this value as background signal from every measuring point. Finally, phosphorylation signals were normalized to the total receptor (phosphorylation-independent antibody; NOPR). Controls (MOCK or SCR) were defined as 100% and phosphorylation of every target protein was calculated as percentage phosphorylation in comparison to the respective control. Statistical analysis was carried out with two-way ANOVA followed by Bonferroni correction. P values <0.05 were considered statistically significant.

To compare the ability of different agonists to induce NOP-YFP internalization in primary cultures of ventral midbrain neurons, fluorescent puncta formation was used as a proxy for receptor internalization. This allowed analysis to be performed on images containing several neurons with many of overlapping neurites. It also allowed automated analysis over large numbers of images without the need to manually select regions corresponding to the plasma membrane and cell interior. Puncta identification and counting was performed using Python code that was based on the original approach and Matlab code developed by Aguet et al. 2013 (128). In order to quantitatively compare the extent of puncta formation across different conditions, the number of puncta per image was normalized to the total NOP-YFP intensity. The plot shows this “puncta density” for each condition relative to N/OFQ.

Supplementary Material

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Figure S1: NOP receptor phosphorylation is mediated by GRK2 and GRK3.

Figure S2: Correlation between NOP receptor phosphorylation and internalization.

Figure S3: Clinically relevant opioids as well as MOP-, DOP- and KOP receptor-selective agonists failed to induce NOP receptor phosphorylation.

Figure S4: Establishment of the membrane potential assay.

Figure S5: Quantification of agonist-induced NOP receptor phosphorylation in mouse brain.